The mammalian gut is provided with specialized tissues of the immune system that serve several related functions. A principal tissue carrying out these functions is the Peyer's patches, specialized sites facing the lumen of the gut. Peyer's patches sample the contents of the lumen, process antigen, and develop an immune response. This response can lead to either cell mediated or humoral immunity, or it can lead to immunologic tolerance. The type of immune response that develops depends upon how the antigen is processed and the current physiological milieu within the Peyer's patches. Analogous immune processes are believed to occur in the airways involved in breathing.
In a fashion analogous to the development of tolerance by the mucosal immune tissues to ingested antigen, early in life the immune system of an animal also develops tolerance to the macromolecules within its own body that could otherwise serve as potential immunogens. The immune system recognizes as "self" the constituents of the body that normally reside therein, and eliminates those of its components that, if not eliminated, would react immunologically against these potential self immunogens. In certain pathological instances, however, the immune system subsequently develops an immunologic response against a self immunogen. This may occur at any time in the life of an animal. In such an event, the components of the immune system react in an immunospecific fashion with the self immunogen, or autoimmunogen, leading to any of a variety of pathological conditions. Examples include multiple sclerosis, in which the self immunogen is myelin or a component thereof, uveitis, in which the self immunogen is the S antigen, a component of the retina; rheumatoid arthritis, in which the self immunogen is one of the classes of collagen which occur in cartilage at the locus of joints; and so on. In such cases, naturally occurring components that were once recognized as "self" become treated as "foreign."
The immunological mechanisms associated with oral tolerization may be classified as active suppression, clonal anergy, and clonal deletion. In general, depending on conditions, these mechanisms may operate independently or in combination. The distinction between these mechanisms depends primarily on the dose of antigen detected by the mucosal tissues, and by the incidence of dosing. Active suppression occurs with repetitive low doses of antigen, in which the result is induction of regulatory T.sub.h2 and T.sub.h3 cells. These cells, when restimulated by the autoantigen, secrete cytokines such as transforming growth factor (TGF)-beta, interleukin (IL)-4, and IL-10, which act to suppress inflammatory responses. Clonal anergy may result with high doses of antigen. A state of unresponsiveness is induced in T.sub.h1 cells, whereby their T cell receptors become incapable of responding to the specific antigen, when processed and presented by the major histocompatibility complex on an antigen presenting cell, that would normally trigger their activation. Clonal deletion, in contrast, occurs upon a single administration of a very high dose of the autoantigen, and results in actual destruction of the T cell clones bearing the specific T cell receptor responsive to the processed antigen. In the presence of high concentrations of antigen in the gut, it is believed that T cells specific for that antigen are eliminated, both within the Peyer's patch itself as well as in the thymus gland.
In recent years a therapeutic approach to the treatment of autoimmune diseases has been the development of tolerance by means of oral administration of the autoimmunogen related to the disease (Weiner, Annu. Rev. Med. 48: 341-351 (1997)). Multiple sclerosis is thought to result from T cells that recognize myelin components of the central nervous system and incite an immune response. Major components of myelin include myelin basic protein and proteolipid protein. Weiner et al. (Science 259: 1321-1324 (1993)) report that multiple sclerosis patients who were fed daily capsules of bovine myelin had a decreased incidence of attacks, compared to patients in a control group. The number of T cells reactive to myelin basic protein were reduced in the patients that were fed myelin. The dose of bovine myelin was 300 mg given daily for a period of one year. In a followup study using some of the same patients (Hohol et al., Annals N.Y. Acad. Sci. 778: 243-250 (1996)), the same dosing regime was followed for a total of three years. In some female patients a dose of 30 mg daily was pursued for one year without any apparent benefit; these patients were subsequently restored to the 300 mg/day dose. Hohol et al. also disclose a planned two-year multi-center Phase III trial which will involve the same 300 mg/day dose of bovine myelin. Such an extended period of dosing may be necessary to discern any benefit in such a chronic disease, in which patients suffer attacks relatively infrequently.
Weiner (1997) reports several clinical trials of oral tolerance at various stages of progress for treatment of various autoimmune diseases. U.S. Pat. No. 5,643,868 to Weiner et al. discloses administration of insulin by non-parenteral routes as a means for preventing or minimizing Type I, or insulin-dependent, diabetes mellitus in a subject. Oral or enteric formulations are disclosed containing between about 1 mg to about 1000 mg insulin per dose. In non-obese diabetic mice, experimental feeding regimes were, for example, twice weekly for 4 1/2weeks, then once a week for 33 weeks. In such animals, diabetes was prevented at 1 mg doses, but not when the dose was reduced to one-tenth or one-hundredth of that amount.
U.S. Pat. No. 5,651,993 to Edelson et al. discloses pharmaceutical compositions and methods of altering the immune response of a mammal. The pharmaceutical compositions contain antigens related to the particular medical state or pathology for which the immune response was intended to be altered. Administration of such compositions stimulates the antigen presenting cells. The methods entail extracorporeal treatment of the resulting expanded population of antigen presenting cells with the antigen in order to maximize the expression of major histocompatibility complex molecules bound with the antigen-derived peptides. The goal was to immunize patients against malignant cells or lymphocytes responsible for autoimmune disease.
U.S. Pat. Nos. 5,571,500, 5,641,474, and 5,645,820 to Hafler et al. also disclose methods for preventing or treating autoimmune diseases in mammals. An autoantigen specific for the autoimmune disease to be prevented or treated is administered by means of an aerosol composition containing the autoantigen. The autoantigen may include or be replaced by autoimmune suppressive fragments of the autoantigen. Autoimmune diseases treated include multiple sclerosis, rheumatoid arthritis, and others. In experimental models using rats, dosages were delivered either orally or by aerosol over periods ranging from 10-31 days before an immunization that induced an autoimmune condition, to 4 days after the immunization.
Oral tolerance to experimental autoimmune encephalomyelitis (EAE) can be induced in rats by the feeding of myelin. EAE is an animal model of inflammatory demyelinating disease which is considered to be similar to multiple sclerosis. Chronic relapsing EAE was induced in mice by immunizing with murine spinal cord (Al-Sabbagh et al., J. Neurosci. Res. 45: 424-429 (1996)). After the onset of symptoms, groups of mice were orally administered bovine myelin in doses of 1, 10, and 20 mg, respectively, three times weekly for six months. Histological examination showed treated animals had diminished inflammatory and demyelinating foci in the brain and spinal cord after prolonged oral administration of myelin. A milder manifestation of clinical disease was observed in the mice treated with 10 or 20 mg as compared to controls.
In oral tolerance experiments on EAE conducted in animals, myelin basic protein (MBP), a major component of myelin, was fed prior to injection of MBP in order to induce the disease. On examining the antibody isotypes from different lymphoid tissues in such animals, Kelly et al. (J. Neuroimmunol. 66: 77-84 (1996)) found that anti-MBP antibodies change isotype distribution most strongly in mucosal lymphoid tissues (i. e., the Peyer's patches). There was a selective reduction in anti-MBP IgA, but not in IgM.
Immune mechanisms contribute to cerebral ischemic injury. Inflammation plays a role in propagating the damage induced by cerebral ischemia. Thus stroke victims are considered to be candidates for various interventions that modulate immune system responses. Immunosuppressive agents that minimize inflammatory responses improve the outcome in experimental models of stroke. Most available immunosuppressive agents, however, have detrimental systemic side effects that limit their therapeutic use in stroke.
Molecular and cellular mechanisms underlying ischemia in the brain were investigated by Mandai et al. (Neurosci. 77 (3): 849-861 (1997)). In situ hybridization for the mRNA encoding proteolipid protein (PLP), a major component of myelin, showed attenuation of the mRNA 12 hours after middle coronary artery occlusion, and barely detectable amounts of the mRNA at 24 hours. In the ischemic core, immunohistochemistry showed that microtubule associated protein 2 disappeared, and that extensive extravasation of albumin across the blood-brain barrier had occurred. These results show extensive damage to oligodendrocytes after ischemic neuronal damage, and significant breakdown of the blood-brain barrier. In contrast, myelin basic protein was detected as long as five days after ischemia.
An immunogenic response in acute ischemic cerebrovascular diseases has been found to involve expansion of the population of T cells reactive with myelin basic protein, peptides fragments of myelin basic protein, and proteolipid protein (Wang et al., Clin. Exp. Immunol. 88: 157-162 (1992)). The T cell group studied was that which secreted interferon-gamma. Patients suffering acute ischemic cerebral infarction had a far higher number of such T cells than did patients with inflammatory neurological diseases such as aseptic meningitis, aseptic encephalitis, or other neurological diseases. In evaluating these results in relation to analogous studies on multiple sclerosis patients, Wang et al. found the origin and role of the immune responses leading to the presence of autoreactive T cells specific for MBP, MBP peptides and PLP to be unclear.
From the above summary, therefore, it is seen that treatments for stroke and related acute cerebral ischemic injury that specifically diminish the immunological response to the ischemic injury, especially ones that reduce the inflammatory response, are not available. This response involves, in particular, neutrophils, T-cells, and natural killer cells. Methods of treating cerebral ischemic injury that specifically target the infarcted area but do not significantly affect other organs in the patient are also not available. The methods currently available have significant systemic side effects when given to a patient.
Other origins of cerebral infarction are also known. Hemorrhagic stroke, and trauma that produces contusion, for example, lead to extended or focal hematomas which can result in inflammatory responses such as edema. Immune-mediated injury likewise can arise following infections such as herpes simplex encephalitis and cytomegalovirus infection.
In the treatment of cerebral ischemic injury (e.g., stroke), and of infarction originating from other causes, there remains a need for methods to minimize or eliminate the immunologic response to substances in the brain that are normally sequestered from the immune system. Breakdown of the blood-brain barrier, to the extent that it occurs, allows exposure of such substances to the immune cells in the circulatory system and facilitates an autoimmune response, including inflammation, to brain tissue, which is absent under normal circumstances. Thus, there remains a need to reduce the severity and extent of such an inflammatory response. There also remains a need for a method of treatment that specifically targets the region of the ischemic injury. Furthermore, there is a need for a method of treatment which does not carry with it the potential for systemic side effects. The present invention satisfies these needs.